Project Summary Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine malignancy in the skin. A newly discovered human polyomavirus, Merkel cell polyomavirus (MCV), encodes oncogenic small and large T antigens that are found in more than 80% of MCC. MCV T antigens promote transformation of normal cells and are required for MCC maintenance. But the precise molecular mechanism by which these viral proteins accomplish this has yet to be determined. Studying MCV T antigens will provide a unique opportunity to investigate the pathogenesis of MCC and other aggressive neuroendocrine cancers and to discover much needed therapeutic targets. Recently, the DeCaprio laboratory discovered that MCV Small T antigen (ST) specifically associates with MYCL (L-MYC) transcription factor and the EP400 chromatin remodeler complex to activate gene expression. We found that ST in a complex with EP400 and MYCL activates the Merkel cell- specific transcription factor ATOH1, as well as components of the coREST transcription repressor complex. coREST inactivation using chemical inhibitors in MCC causes a growth arrest and an increase in Notch tumor suppressor signaling genes. Here we hypothesize that through direct activation and indirect repression of Notch signaling genes via coREST, ST increases ATOH1 in MCC to promote oncogenesis. We also found that coREST inactivation upregulates the LGR6 skin stem cell receptor. In vivo LGR6 expression is restricted normally to hair follicle stem cells. Recent work in an LGR6 lineage-tracing mouse model suggests that LGR6+ adult stem cells are able to reconstitute the entire interfollicular epithelium after wounding, suggesting the possibility that MCC displaying Merkel cell properties may arise from these cells. Thus, we propose the following specific aims to test the hypothesis that Merkel cell polyomavirus causes cancer by activating ATOH1 and inhibiting Notch in hair follicle stem cells. In Aim 1, I will determine whether ST induces ATOH1 through direct activation EP400-MYCL and Notch signaling gene repression by coREST. I will investigate ST?s occupancy and the effect of coREST on Notch signaling gene expression in MCC cell lines. In Aim 2, I will assess the role of the ATOH1 transcription program and Notch signaling in MCC maintenance. To do so, I will study the ATOH1 transcriptome and probe the effect of Notch signaling activation in MCC. In Aim 3, I propose to determine whether LGR6-driven expression of Merkel cell polyomavirus T antigens in the hair follicle stem cells leads to accumulation of ATOH1+ Merkel cell-like cells in mouse skin. Impact: After completing these aims, the proposed research will uncover the unappreciated role of ATOH1, coREST and Notch signaling pathway in MCC and determine how ST contributes to MCC through modulating host transcription programs. These findings will disclose novel cancer targets for MCC and potentially other difficult to treat neuroendocrine malignancies.